Replication fork instability and the consequences of fork collisions from rereplication

Abstract

Replication forks encounter obstacles that must be repaired or bypassed to complete chromosome duplication before cell division. Proteomic analysis of replication forks suggests that the checkpoint and repair machinery travels with unperturbed forks, implying that they are poised to respond to stalling and collapse. However, impaired fork progression still generates aberrations, including repeat copy number instability and chromosome rearrangements. Deregulated origin firing also causes fork instability if a newer fork collides with an older one, generating double-strand breaks (DSBs) and partially rereplicated DNA. Current evidence suggests that multiple mechanisms are used to repair rereplication damage, yet these can have deleterious consequences for genome integrity.

Keywords: break-induced replication; double-strand break repair; gene amplification; homologous recombination; nonhomologous end-joining.

Figure 1.

Summary of the eukaryotic DNA replication fork. Cdc45, the Mcm2–7 complex, and the GINS complex comprise the CMG helicase that unwinds the dsDNA. Leading strand synthesis is shown at the top and is accomplished by Pol ε. Lagging strand synthesis is depicted below. Pol α primase synthesizes 8- to 15-nucleotide-long RNA primers along the lagging strand. Synthesis of the lagging strand is performed by Pol δ. PCNA binds to Pol δ and Pol ε to enhance processivity. The nucleosome remodelers FACT and ASF1 bind to Mcm2–7 to coordinate removal of nucleosomes with the oncoming replication fork. ASF1 also cooperates with CAF-1 to deposit new H3–H4 tetramers behind the elongating fork. (Illustration by Steven Lee, http://www.graphiko.com. Adapted by permission from Macmillan Publishers Ltd: Nature Reviews Molecular Cell Biology [Alabert and Groth 2012] © 2012.)

Figure 2.

Pathways of DSB repair. Resection of the DSB commits repair to HR or alternative end-joining (alt-EJ) repair pathways. (Left) Alt-EJ by microhomology-mediated end-joining (MMEJ) requires Pol θ to align or template short microhomologies, generating deletions and insertions. The ends are then ligated together by the Ligase III (Lig3)/XRCC1 complex. (Middle) HR repair requires BRCA2 to recruit Rad51 and facilitates filament formation along the resected DNA. Rad51 filaments search for homologous sequences and initiate strand invasion to restore the exact sequence to the break site. (Right) Resection is blocked in nonhomologous end-joining (NHEJ) by Ku70–80 binding. The Ku70–80 heterodimer recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and this complex brings the broken DNA ends together. The XRCC4–DNA Ligase IV (Lig4) complex is recruited and catalyzes DSB ligation. (Illustration by Steven Lee, http://www.graphiko.com.)

Figure 3.

The fork collision model of DSB generation by rereplication. (A) Collisions between two replication forks on the same DNA duplex (green) would generate a DSB behind the second replication fork (arrows). Multiple origin reinitiations would increase the frequency of fork collisions and thus the number DSBs formed. Fork collisions are expected to be stochastic and may occur at only a subset of forks, as shown here. (B) One proposed mechanism of DSB formation. If the leading strand (red) from a second fork collides with unligated Okazaki fragments on the lagging strand of an earlier fork (blue), a DSB results. Note that, although this is single-end DSB, fork collisions on opposite sides of the origin could generate two ends that could be joined by NHEJ. Other mechanisms such as exonuclease cleavage or steric breakage of the DNA also could generate additional DSB ends. (Illustration by Steven Lee, http://www.graphiko.com.)